Bacteria antigen for the treatment of scarlet fever



Patented May 23, 1 944 BACTERIA ANTIGEN FOR THE TREATMENT OF SCARLETFEVER Orry Charles Morrison, Carroll, Iowa No Drawing. Application May2,1938, Serial No. 205,624

7 Claims.

This invention relates to a bacteria antigen for the treatment ofscarlet fever, a method of preparing the same, and a method of treatmentfor I and immunizing against scarlet fever.

This application is a continuation-in-part of my copending applicationSerial No. 9,914 filed March 7, 1935, which was a continuation-impart ofmy then copending application Serial No. 510,480 filed January 23, 1931.

In preparing the antigen, the Streptococcus scarlatinae is grown in massculture on a media of beef infusion agar containing one percent ofdextrose and fifteen percent sterile defibrinated beef blood. The bloodis added after the media has been sterilized. The media is then testedfor sterility by incubating at 37.5 C. for 24 hours.

The cultures used were obtained from Parke Davis and Co., and the strainwas kept up by transplanting the cultures twice a week on blood agar.The usual microscopic examinations and morphological tests were madebefore transplanting to mass culture bottles. 1 cubic centimeter of thegrowth is produced by an inoculation into a beef infusion broth, thentransferred onto each of the blood agar slants in mass culture bottles.If any evidence of contamination appears, the bottle is carefullychecked and discarded if not pure.

When the growth is judged to be sufllcient, which is usually after about36 hours, the bacteria are washed oif the media by means of sterilesaline solution and by gently rubbing the slant surface with a wiretriangle. The emulsion of bacteria is then removed with a sterilepipette and centrifuged in a sterile tube. The bacteria are then washedfive times or more in physiological sterile saline solution until thereis no culture media present.

The bacteria are then removed and placed in a dryer at 56 C., in whichthey are all killed by the heat. The time necessary for drying isdetermined by the quantity of bacteria present.

When the material appears to be dry, it is weighed and then returned tothe oven for 24 hours and then reweighed. If the weight remains the sameas the day before, the material is then ready for treating in a ballmill.

The sterile physiological saline solution is added to the dried bacteriaafter they have been weighed so that there will be 1000 cc. of salinesolution for each gram of dried bacteria. The mixture is now placed inthe ball mill. A suitable preservative, preferably phenol, is added. Ifphenol is used, the percentage is 0.5%. The jar is properly corked andthen turned at the speed of 70 revolutions per minute for a period of 60hours. The material is then removed from the ball mill and passedthrough a fine Berkefeldt filter, in which all parts of bacteria orbacterial bodies are removed. If the grinding has been suflicient, therenormally willnot be any whole bacterial'bodies.

The filtrate is tested for sterility by inoculating 1 cubic centimeterinto a Smithe fermentation tube. Normally three tubes are employed andthese are incubated for seven days. If the filtrate is found to besterile, it is then bottled into sterile bottles and a sample taken atrandom from three of these bottles and tested. The tests should be atthe rate ofat least 2% of the bottles made up. Should these be found tobe sterile, then the antigen is ready for use. If any of them are notsterile, the entire amount is refiltered and the steps repeated.

The antigen is stored in a refrigerator at 40 F. awaiting use.

The heat treatment of the bacteria, particularly while moist, results inthe precipitation of the protein content thereof, and in the subsequentcentrifugatlon'and filtration, all protein matter is removed. As aresult, the material is non-toxic and. non-irritant and extraordinarilylarge doses of it may be employed without undesirable aftereffects.

The dosage of material normally employed is l cc. of antigen giveneither subcutaneously or intravenously once each day. Much largerquantities, however, may be employed in immunizing from scarlet fever.The preferred practice is first to determine susceptibility by the Dicktest and then to administer antigen at the rate of 1 cc.

per day for five successive days, after which susceptibility is againtested and if the patient is again susceptible, the injections arecontinued until the test is negative.

The antigen may likewise be used after the onset of the disease in thesame manner, although in such cases larger dosages may be desirable.

While the chemical and physiological aspects of the antigen are notcompletely understood, it is believed that the antigen comprises inlarge part material antipathetic to the bacterial enzymes.

The scarlet fever bacteria exude enzymeswhich act upon the bodilytissues to digest. the same and render them assimilable by the bacteria.bacteria itself apparently contains a large excess of antipatheticmaterial which protects the bacteria itself from solution or digestion.These anti-enzymes which act to build up the tissues rather than breakthem down, or at least prevent the breaking down of the tissues, will becalled herein genzymes. The introduction of such ma.- terial into thebodily tissues will naturally prevent the digestion thereof by thecorresponding The clearness of understanding only, and no unnecessarylimitations should be understood therefrom,

but the appended claims should be construed as broadly as permissible inview of the prior art.

I'claim:

1. The method of producing immunizing materialwhich comprisessegregating a substantially pure culture oi -Streptococcus scarlatinae,multiplying the culture upon a protein medium, separating the resultingbacterial growth from the culture medium, subjecting the bacteria to atemperature of the order of 56 C.'high enough to destroy them and toprecipitate all protein matter therein, comrninuting the bacteria, andremoving all solid matter from the resulting ground material.

2. The method'as set forth in claim 1 in which the bacteria arecomminuted in the presence of a predetermined proportion ofphysiological salt solution.

3. The method of producing immunizing material which comprisessegregating a substantially pure culture of Streptococcus 'scarlatz'nae,multiplying the culture upon a protein medium,separating the resultingbacterial growth from the culture medium, subjecting the bacteria to atemperature of the order of 56 C. high enough to destroy them and toprecipitate all protein matter therein, drying and weighing the bacteriaand adding a predetermined proportion of saline solution thereto,comminuting the bacteria, and removing all solid matter from theresulting ground material.

4. The method as set forth in claim 1 in which the bacteria arecomminuted in the presence of an excess of physiological salt solution.

5. A scarlet fever immunizing preparation comprising a bacterial extractproduced according to the method of claim 1.

6. A Streptococcus carlatinae immunizing agent of predetermined strengthand prepared according to the process of claim 3.

'7. A Streptococcus scarlatinae immunizing agent of predeterminedstrength and prepared according to the process of claim 3 and in whichthe ratio of dried bacteria to saline solution is approximately 1 gramof bacteria per 1,000 cos. of solution.

ORRY CHARLES MORRISON.

